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  • Höijer, IdaUppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik (author)

Amplification-free long-read sequencing reveals unforeseen CRISPR-Cas9 off-target activity

  • Article/chapterEnglish2020

Publisher, publication year, extent ...

  • 2020-12-01
  • Springer Science and Business Media LLC,2020
  • electronicrdacarrier

Numbers

  • LIBRIS-ID:oai:DiVA.org:uu-428344
  • https://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-428344URI
  • https://doi.org/10.1186/s13059-020-02206-wDOI

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  • Language:English
  • Summary in:English

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  • Subject category:ref swepub-contenttype
  • Subject category:art swepub-publicationtype

Notes

  • BACKGROUND: One ongoing concern about CRISPR-Cas9 genome editing is that unspecific guide RNA (gRNA) binding may induce off-target mutations. However, accurate prediction of CRISPR-Cas9 off-target activity is challenging. Here, we present SMRT-OTS and Nano-OTS, two novel, amplification-free, long-read sequencing protocols for detection of gRNA-driven digestion of genomic DNA by Cas9 in vitro.RESULTS: The methods are assessed using the human cell line HEK293, re-sequenced at 18x coverage using highly accurate HiFi SMRT reads. SMRT-OTS and Nano-OTS are first applied to three different gRNAs targeting HEK293 genomic DNA, resulting in a set of 55 high-confidence gRNA cleavage sites identified by both methods. Twenty-five of these sites are not reported by off-target prediction software, either because they contain four or more single nucleotide mismatches or insertion/deletion mismatches, as compared with the human reference. Additional experiments reveal that 85% of Cas9 cleavage sites are also found by other in vitro-based methods and that on- and off-target sites are detectable in gene bodies where short-reads fail to uniquely align. Even though SMRT-OTS and Nano-OTS identify several sites with previously validated off-target editing activity in cells, our own CRISPR-Cas9 editing experiments in human fibroblasts do not give rise to detectable off-target mutations at the in vitro-predicted sites. However, indel and structural variation events are enriched at the on-target sites.CONCLUSIONS: Amplification-free long-read sequencing reveals Cas9 cleavage sites in vitro that would have been difficult to predict using computational tools, including in dark genomic regions inaccessible by short-read sequencing.

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  • Johansson, JosefinUppsala universitet,Medicinsk genetik och genomik,Science for Life Laboratory, SciLifeLab(Swepub:uu)josjo523 (author)
  • Gudmundsson, Sanna,1989-Uppsala universitet,Medicinsk genetik och genomik,Science for Life Laboratory, SciLifeLab,Program in Medical and Population Genetics, Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA; Division of Genetics and Genomics, Boston Children’s Hospital, Boston, MA, USA(Swepub:uu)sangu463 (author)
  • Chin, Chen-Shan (author)
  • Bunikis, IgnasUppsala universitet,Science for Life Laboratory, SciLifeLab,Institutionen för immunologi, genetik och patologi(Swepub:uu)ignbu776 (author)
  • Häggqvist, SusanaUppsala universitet,Institutionen för immunologi, genetik och patologi,Science for Life Laboratory, SciLifeLab(Swepub:uu)susha415 (author)
  • Emmanouilidou, AnastasiaUppsala universitet,Medicinsk genetik och genomik,Science for Life Laboratory, SciLifeLab(Swepub:uu)anaem996 (author)
  • Wilbe, MariaUppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik(Swepub:uu)marwi726 (author)
  • den Hoed, Marcel,1980-Uppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik(Swepub:uu)marde358 (author)
  • Bondeson, Marie-Louise,1960-Uppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik(Swepub:uu)malobond (author)
  • Feuk, LarsUppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik(Swepub:uu)larsfeuk (author)
  • Gyllensten, UlfUppsala universitet,Science for Life Laboratory, SciLifeLab,Medicinsk genetik och genomik(Swepub:uu)ulfgyll (author)
  • Ameur, AdamUppsala universitet,Institutionen för immunologi, genetik och patologi,Science for Life Laboratory, SciLifeLab,Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Australia(Swepub:uu)adame789 (author)
  • Uppsala universitetScience for Life Laboratory, SciLifeLab (creator_code:org_t)

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  • In:Genome Biology: Springer Science and Business Media LLC21:11465-69061474-760X

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